The present invention relates to methods for the diagnosis of allergic bronchopulmonary aspergillosis (ABPA) and recombinant allergens to be used in the methods. During the priority year the recombinant allergen that in the priority application was called rAsp f2 has officially been named rAsp f6. The official name is used in this text.
Allergic bronchopulmonary aspergillosis (ABPA). Allergic bronchopulmonary aspergillosis is the most severe allergic complication caused by Aspergillus species, mainly A. fumigatus. ABPA is the result of hypersensitivity to Aspergillus-antigens mainly in patients suffering from long-standing atopic asthma (8-12) or cystic fibrosis (16-19). Although originally considered as a rare disease (13), ABPA is currently recognized with much greater frequency. ABPA with varied clinical presentations has been reported to occur in about 15% of the asthmatic patients sensitized to A. fumigatus (14,15), while in patients with cystic fibrosis the reported incidence varies from 10 to 35% (16,17). ABPA has been described as an immune disease that ranges from asthma to fatal destructive lung disease with defined clinical, serological, radiological and pathological features (8,18-22). Because of its severity ABPA should be ruled out in patients with chronic asthma or cystic fibrosis exhibiting immediate cutaneous reactivity to A. fumigatus (8). The diagnostic criteria for ABPA are asthma or cystic fibrosis, history of roentgenographic infiltrates (in most cases), immediate cutaneous reactivity to A. fumigatus extracts, elevated total serum IgE, precipitating antibodies to A. fumigatus, peripheral blood eosinophilia, elevated specific serum IgE and IgG to A. fumigatus as compared to sera from patients with asthma and cutaneous reactivity to Aspergillus, but without ABPA, and proximal (central) bronchiectasis with normal tapering of distal bronchi (23-25). In cases where all criteria are present, diagnosis is readily made (26). However, all of the eight criteria are rarely present at the same time even in classic ABPA-patients with central bronchiectasis. With exception of bronchiectasis and to some extent elevated specific serum IgE and IgG to A. fumigatus, none of the diagnostic criteria are specific for ABPA (26). Furthermore, pulmonary infiltrates and central bronchiectasis are commonly detected in patients suffering from cystic fibrosis also in the absence of sensitization to A. fumigatus, which makes a diagnosis of ABPA in patients with cystic fibrosis even more difficult (16). Therefore, serologic identification of ABPA has a greater diagnostic potential, but is, however, hampered by the lack of standardized, reliable fungal extracts (5,7,27-29).
Aspergillus fumigatus antigens. The major problem in the immunodiagnosis of diseases related to A. fumigatus stems from the antigenic complexity of the fungus. Antigen/allergen extracts of A. fumigatus contain hundreds of different proteins (6,30,31), of which a limited subset are able to let bind human serum IgE (6,32,33,35). The fungus has been reported to produce more than 40 IgE-binding components which generate complex IgE-binding patterns when extracts are examined by Western blot analysis using sera from allergic individuals (32,33). To make the picture even more complicated, serum IgE from different patients recognize highly variable patterns of fungal proteins (6,36). In the case of patients suffering from ABPA, depending on the stage of the disease, different allergenic xe2x80x9cfingerprintsxe2x80x9d may be obtained with serum of the same patient taken at different times, even if fungal extract from the same batch is used (36,37).
It has been suggested to use purified native allergenic components instead of crude allergen extracts for diagnosing ABPA (79). Recombinant A. fumigatus allergens with connections to ABPA have been described earlier (71,83).
The inventors are named authors in a number of articles about recombinant allergens from A. fumigatus (cloning and expression: 39,43,49,51,52,82, and diagnostic use: 59,66,32,71,76,81).
Result of International-Type Search During the Priority Year.
The references 66, 79 and 84 have been categorised as being of particular relevance.
Banerjee et al., (84) describes antigens that cannot be intracellular. The described antigens are shown to react with sera of patients with ABPA, but there are no data suggesting that the antigens will not react with sera of A. fumigatus-sensitised patients not having ABPA.
Moser et al (66) and Little et al., (79) describe secreted proteins/antigens that do not allow for differential diagnosis of ABPA because they frequently reacts with sera of A. fumigatus-sensitised patients without ABPA and sera of ABPA patients.
The main objective of the invention is to provide improved methods for diagnosis of ABPA.
One subobjective is to provide in vitro diagnostic methods that have the sufficient specificity and sensitivity for diagnosis of ABPA.
A second subobjective is to provide well-defined allergen preparations that can be used for the diagnosis of ABPA both in vitro and in vivo, including immunoassay and skin reactivity measurement methods, respectively.